Comparison of Gelation Time and Polyalcohol Effect on Hydrogels from Domestic and Wild Silk Fibroins

Zhao, Huijing; Xiong, Siyong; Li, Mingzhong; Zhang, Qiang; Liu, Guiyang

doi:https://doi.org/10.1155/2012/819464

Advances in Materials Science and Engineering

On this page

Abstract Introduction Materials and Methods Results and Discussion Conclusions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2012 | Article ID 819464 | https://doi.org/10.1155/2012/819464

Comparison of Gelation Time and Polyalcohol Effect on Hydrogels from Domestic and Wild Silk Fibroins

Huijing Zhao,¹Siyong Xiong,²Mingzhong Li,¹Qiang Zhang,¹and Guiyang Liu^1,3

Academic Editor: Wen-Hua Sun

Received20 Aug 2011

Revised20 Jan 2012

Accepted08 Feb 2012

Published08 Apr 2012

Abstract

Silk fibroin (SF) hydrogels were obtained from both domestic (Bombyx mori) and wild (Antheraea pernyi) silkworms from aqueous silk fibroin solutions at room temperature. The gelation time of the Antheraea pernyi (A. pernyi) SF solution was significantly shorter than that of the Bombyx mori (B. mori) SF solution. The secondary structures of the two kinds of hydrogels were also compared. In order to further reduce the gelation time, various amounts of polyethylene glycol (PEG) were blended with the silk fibroins of A. pernyi and B. mori. The gelation time of both A. pernyi SF and B. mori SF decreased with the increased amount of PEG. After freeze-drying, the hydrogels were characterized through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Results showed that the addition of polyalcohol did not change the main secondary structure of the hydrogels. However, the addition of polyalcohol did reduce the gelation time and triggered additional formation of β-sheets.

1. Introduction

Hydrogels can maintain a distinct three-dimensional porous structure with mechanical and structural properties similar to that of many natural tissues and extracellular matrices (ECMs) and can be delivered in a minimally invasive manner [1]. Therefore, hydrogels are of interest for many biomedical applications such as tissue engineering scaffolds, controlled drug release devices, and biosensors [2]. Hydrogels can be made of either synthetic materials or naturally derived materials [3]. Among naturally derived materials, it is agreed that silkworm silk fibroin (SF) is one of the most promising biomaterials because of its excellent biocompatibility, biosafety, controllable biodegradation rates, processability, and mechanical properties [4]. Silkworm silk fibroin is excreted by domestic (B. mori) and wild (A. pernyi) silkworms. Domestic SF hydrogels are of interest for use as bone-filling biomaterials, wound dressing, and so on and have proved to be promising for healing efficacy [5–10]. Compared with B. mori SF, A. pernyi SF, the most familiar wild SF, has more advantages in terms of chemical activity and high-affinity interactions with mammalian cells [11]. A. pernyi SF is rich in amino acids having polarity and ionogenic pendant groups, which are the potential reactive sites of chemical reactions. In terms of amino acids, A. pernyi SF has more Ala, Asp, and Arg and less Gly than does B. mori. Further, A. pernyi SF also has abundant alkaline amino acids (Arg and His) and the tripeptide sequence Arg-Gly-Asp (RGD), which is known to be a cell integrin receptor and to mediate special interactions between mammalian cells. It has been demonstrated in some research that A. pernyi SF performs better in terms of cell adhesion, growth, and phenotypic maintenance than does B. mori SF [12]. Also, A. pernyi has bioproperties similar to collagen, such as cell adhesion, growth, and proliferation. Therefore, it is supposed that biomedical materials made of A. pernyi SF have better bioproperties than do those made of B. mori SF. However, biomaterials made of A. pernyi SF have not been studied as widely as those made of B. mori SF, especially in hydrogels. Because of their improved properties and better performance, introducing wild SF hydrogels as a potential biomaterial in tissue engineering is very necessary. Therefore, hydrogels made of A. pernyi SF were prepared and studied in the present study.

Through reviewing research articles about B. mori SF hydrogels, we found that the gelation time of SF hydrogels is excessively long unless nonphysiological treatments (such as low pH, high temperature, or additives) are considered [6, 13–17]. The question we want to answer is do the A. pernyi SF hydrogels have a similar problem? This question prompted us to search for effective ways to reduce the gelation time of A. pernyi SF solutions. In addition to the methods of γ-ray irradiation, ultrasonication, and shearing, blending auxiliary materials like polyalcohol with SF has been considered an effective method to reduce gelation time because it induces the transition of SF from random coil and α-helix structures to β-sheet structures [18]. Among polyalcohols, polyethylene glycol (PEG) is a linear polymeric material that is polymerized by glycol monomers. The presence of abundant oxethyl groups in the PEG molecule that are capable of forming hydrogen bonds with water makes PEG a highly water-soluble material. PEG is also a nontoxic, nonimmunogenic, and amphipathic polymer, and it possesses a variety of properties pertinent to biomedical and biotechnical applications [18–20]. Thus, adding PEG to A. pernyi SF solutions was selected as a method for reducing gelation time in this study. B. mori SF hydrogels with and without PEG were also prepared as control samples. A. pernyi and B. mori SF hydrogels were characterized after freeze-drying through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy. The effect of PEG on gelation time and the final secondary structure of the hydrogels were also studied.

2. Materials and Methods

2.1. Preparation of SF Aqueous Solutions

The process of preparing SF aqueous solution is described elsewhere [11, 21]. To be specific, cocoons of A. pernyi were boiled for 30 min in a 0.25 wt% Na₂CO₃ aqueous solution to remove sericin. Degummed A. pernyi SF fibers were rinsed thoroughly with pure water. After oven drying (60°C), they were dissolved in Ca(NO₃)₂·4H₂O (the liquor ratio was 1 : 10) at 105°C for 5 h. Then, the mixed solution was dialyzed against flowing distilled water for 96 h to remove salt. The concentration of A. pernyi SF aqueous solution was about 1.78 wt%, and it was then concentrated to 3.0 wt% through air drying.

B. mori silks were degummed by being boiled for 30 min in a 0.06 wt% Na₂CO₃ aqueous solution. After being rinsed and oven dried (60°C), the degummed B. mori SF fibers were dissolved in the triadic solvent CaCl₂·CH₃CH₂OH·H₂O (mole ratio = 1 : 2 : 8) at 72°C for 1 h with stirring; the liquor ratio of the triadic solvent was 1 : 10. This solution was dialyzed against flowing distilled water for 96 h. The final concentration of B. mori SF solution was also about 3.0 wt%.

2.2. Preparation of SF Hydrogels

PEG 600 was added to the two 3.0 wt% SF solutions in ratios of 0, 50, 100, 150, 300, 500, 700, and 900 wt% (PEG/SF) and gently stirred at room temperature to disperse the PEG homogeneously. The mixed solutions were then left to stand at room temperature. The formation of gels was determined by inserting a glass capillary into the mixed solutions; when the liquid level in the glass capillary stopped rising, the gelation time of each sample was recorded. In order to measure their structure, the hydrogels were then frozen at −40°C and lyophilized.

2.3. X-Ray Diffraction (XRD)

The hydrogel samples were made into powders and measured by X-ray diffractometer (X’PERT PRO MPD) with Cu Kα radiation (λ = 1.5406 Å). The scanning speed was 2°/mm. The diffraction intensity curves with 2θ from 5 to 45° were obtained.

2.4. Fourier Transform-Infrared (FT-IR) Spectroscopy

The hydrogel samples were made into powders, using KBr tabletting to prepare specimens. An FT-IR spectrophotometer (Nicolet Avatar-IR 360) was used to analyze the samples. All spectra were recorded by absorption mode at 2 cm⁻¹ interval and in the wavelength range of 1800~400 cm⁻¹.

2.5. Raman Spectroscopy

Raman spectra were obtained by Raman spectrometer (LabRam-1B) at a wavelength of 632.8 nm, the resolution of which was 2 cm⁻¹. The scanning time for solid specimens was 200 s.

3. Results and Discussion

3.1. Gelation Time

It can be seen in Figures 1 and 2 that the original gelation time of A. pernyi SF was 69.3 hours, which is much shorter than that of B. mori SF (gelation time was 288.6 h). Pure A. pernyi SF can also gelate quicker than that of B. mori SF with 500 wt% PEG (gelation time is 103.1 h). The structure of the SF hydrogels was stabilized due to the formation of β-sheets, which served as physical cross-links [22, 23]. A. pernyi SF is rich in amino acids having polarity and ionogenic pendant groups, which more easily forms reactions with other polar molecules and functional groups. The movements and interactions between the SF molecules themselves and the PEG in the A. pernyi SF aqueous solution would be more violent than those in the B. mori SF aqueous solution. The transition of β-sheets would be therefore more rapid and much easier.

The gelation time of A. pernyi SF decreased with the addition of PEG, and it decreased to 38.0 h when the amount of PEG was 500 wt%. A small amount of PEG (only 50%) can reduce the gelation time of A. pernyi SF, whereas the gelation time of B. mori SF required the addition of as much as 300 wt% PEG to significantly reduce the gelation time. PEG was highly water-soluble once it was blended with the SF aqueous solution, forming abundant hydrogen bonds and breaking the hydration layer around the SF molecules. The molecules of SF and PEG started to move and collide with each other, which accelerated the aggregation of SF molecules and triggered the breaking and rebuilding of hydrogen bonds. Through this process, random coil and α-helix structure were induced to transform into β-sheets.

Figure 2 shows that preparing B. mori SF is time-consuming, which is a drawback for the fabrication of SF hydrogels. However, in this study, it was found that A. pernyi SF can gelate much quicker than B. mori SF. Besides, adding a small amount of PEG (only 50%) can further reduce the gelation time of A. pernyi SF. Hydrogels from A. pernyi SF also have better bioproperties and are more advantageous in terms of high-affinity interactions with cells; the shorter preparation time is another advantage for the use of these hydrogels in potential biomedical applications.

3.2. X-Ray Diffraction

The formation of β-sheets is an important issue in stabilizing the structure of SF hydrogels. Secondary conformation of hydrogels from pure A. pernyi SF, pure B. mori SF, and SFs blended with PEG are shown in the XRD curves in Figure 3. For the diffraction peaks of silk fibroin, please see [21, 24, 25]. In Figure 3(a), the diffraction peaks of pure A. pernyi SF hydrogels, shown in curve (A), appear around 16.5°, 20.2°, 24.9°, and 30.9° and are attributed to β-sheet structure. The diffraction peaks at 16.5°, 24.9°, and 30.9° become wider (curve (B)) and then disappear (curve (C)), while the main diffraction peaks at 20.2° remain. These results indicate that the secondary structure of A. pernyi SF hydrogels contains mainly β-sheets; however, with the addition of PEG (100 wt% and 300 wt%), the diffraction peaks of the hydrogels are covered up by those of PEG. A similar phenomenon is observed in Figure 3(b), in which the secondary structure of pure B. mori SF (curve (A), with diffraction peaks at 9.1°, 20.7°, and 24.7°) consists mainly of β-sheets. With the addition of PEG, diffraction peaks at 9.1° and 24.7° are covered up, while peaks at 20.7° remain (curves (B) and (C)). The diffraction curves of the hydrogels with other blending ratios of SF and PEG were the same, so the data for these samples are not shown; only representative curves were selected for analysis. Figure 3 indicates that the secondary structure of both A. pernyi SF and B. mori SF are mainly β-sheets, and the addition of PEG did not influence the crystalline structure of the SF hydrogels.

(a)

(b)

3.3. FT-IR Spectroscopy

In order to prove the results of X-ray diffraction, FT-IR spectra were also obtained in this study, the results of which are shown in Figure 4. For the characteristic peaks of silk fibroin in FT-IR spectra, see [25–28]. In Figure 4(a), relatively strong characteristic peaks appear in curve (A) at 1631 cm⁻¹ (amide I), 1521 cm⁻¹ (amide II), 1240 cm⁻¹ (amide III), 965 cm⁻¹ (amide IV), and 700 cm⁻¹ (amide V), which means that pure A. pernyi SF hydrogels contain large amounts of β-sheets. Meanwhile, weak characteristic peaks appear in curve (A) at 881 cm⁻¹ (amide IV) and 620 cm⁻¹ (amide V), which indicates that a small amount of α-helix structures coexist with the β-sheets. With the addition of PEG, the characteristic peaks attributed to β-sheets become stronger (curves (B) and (C)), while those attributed to α-helix structures become smoother (curves (B) and (C)). A similar phenomenon is observed in Figure 4(b), in which the secondary structure of pure B. mori SF (curve (A)) consists of a large amount of β-sheets (1635 cm⁻¹, 1620 cm⁻¹, 1234 cm⁻¹, and 700 cm⁻¹) and a small amount of α-helix structures (610 cm⁻¹). With the addition of PEG, the characteristic peaks attributed to β-sheets become stronger (curves (B) and (C)), while those of the α-helix (610 cm⁻¹) disappear. The above phenomena indicate that the addition of polyalcohol triggered the transition of more β-sheets and that the main secondary structure in A. pernyi and B. mori SF hydrogels remains β-sheets.

(a)

(b)

3.4. Raman Spectroscopy

In order to confirm the results of X-ray diffraction and FT-IR, laser Raman spectroscopy of A. pernyi and B. mori SF hydrogels with added PEG at ratios of 0 and 300 wt% were detected, as shown in Figure 5. For the characteristic peaks of silk fibroin in Raman spectra, see [29]. In Figure 5(a), strong characteristic peaks in the Raman spectra of pure A. pernyi SF hydrogels (curve (A)) show around 1669.07 cm⁻¹, 1223.62 cm⁻¹, and 1093.78 cm⁻¹, which are attributed to β-sheets, while relatively weak characteristic peaks appear around 1657.88 cm⁻¹, 1646.67 cm⁻¹, 1269.36 cm⁻¹, and 907.29 cm⁻¹, which are attributed to the α-helix. Similarly, the secondary structure of pure B. mori SF hydrogels contains large amounts of β-sheets (strong characteristic peaks at 1665.99 cm⁻¹, 1235.64 cm⁻¹, 1218.68 cm⁻¹, and 1086.51 cm⁻¹) and small amounts of α-helix (relatively weak characteristic peaks at 1267.32 cm⁻¹ and 1101.78 cm⁻¹) and random coil (weak characteristic peaks at 1257.95 cm⁻¹). With the addition of PEG, the characteristic peaks at 1646.67 cm⁻¹ and 1269.36 cm⁻¹ attributed to α-helix disappear, whereas new characteristic peaks attributed to β-sheets (1241.65 cm⁻¹ and 1078.45 cm⁻¹) appear in curve (B) of Figure 5(a). In Figure 5(b) curve (B), the characteristic peaks attributed to α-helix (1267.32 cm⁻¹ and 1101.78 cm⁻¹) and random coil (1257.95 cm⁻¹) disappear with the addition of PEG, while those of the β-sheets (1666.99 cm⁻¹, 1240.30 cm⁻¹, 1086.51 cm⁻¹, 1064.22 cm⁻¹, and 1038.12 cm⁻¹) show in the curve.

(a)

(b)

These results indicate that the main secondary structure in A. pernyi and B. mori SF hydrogels are β-sheets. With the addition of polyalcohol, more β-sheets in the secondary structure of both A. pernyi and B. mori SF hydrogels were formed. This is consistent with the results of X-ray diffraction and FT-IR.

Based on the above results and discussions, we would like to make some further points. Gelation occurs because SF chains tend to aggregate, passing from an amorphous (random coil) or unstable (α-helix) conformation to a more stable structure (β-sheets). The reactive activity of A. pernyi SF is stronger than that of B. mori SF due to its molecular structure. Therefore, the transition to β-sheets in both the pure A. pernyi SF aqueous solution and the solutions blended with PEG would be more rapid and much easier. Gelation of B. mori SF is both time- and energy-consuming, which has also been proven in other research [6, 7, 24]. In the present study, the gelation time of A. pernyi SF was proven to be much shorter than that of B. mori SF, even when nonauxiliary materials were added. Besides, adding even a small amount of PEG could reduce the gelation time. It has been proven that A. pernyi SF has better bioproperties than does B. mori SF. Therefore, A. pernyi SF hydrogels can be a more promising biomaterial.

The results of X-ray diffraction, FT-IR, and Raman spectroscopy indicate that the pure SF hydrogels (both A. pernyi and B. mori) contain mainly β-sheets, coexisting with a small amount of α-helix and random coil structures. With the addition of PEG, the amount of α-helix and random coil present is reduced, while more β-sheets are formed. This is probably because part of the α-helix and random coil structures have been transformed into β-sheets. The addition of PEG also reduces the gelation time, as described previously. This would further indicate that, while the addition of polyalcohol did not influence the main secondary structure of the hydrogels, it did induce a quicker transition to β-sheets and triggered additional formation of β-sheets.

4. Conclusions

Hydrogels from pure A. pernyi SF, pure B. mori SF, and SF blended with PEG were obtained and their secondary structures were studied. The conclusions of this study are as follows. (1) The gelation time of pure A. pernyi SF was significantly shorter than that of B. mori SF. (2) The gelation time of A. pernyi SF can be reduced even more by adding a small amount of PEG. (3) The addition of polyalcohol did not influence the main secondary structure of the hydrogels. However, it did induce a quicker transition to β-sheets and triggered additional formation of β-sheets.

In this study, hydrogels from wild (A. pernyi) SF were successfully prepared in a much shorter time compared to hydrogels prepared from domestic (B. mori) SF. Considering their better bioproperties, A. pernyi SF hydrogels are a promising biomaterial that can be applied in much wider biofields.

Acknowledgments

This work is supported by the National Natural Science Foundation of China (no. 30970714), Nature Science Foundation of Jiangsu Province (no. BK2010252), and Priority Academic Program Development of Jiangsu Higher Education Institutions.

References

J. Kopeček, “Hydrogel biomaterials: a smart future?” Biomaterials, vol. 28, no. 34, pp. 5185–5192, 2007.
View at: Publisher Site | Google Scholar
J. L. Drury and D. J. Mooney, “Hydrogels for tissue engineering: scaffold design variables and applications,” Biomaterials, vol. 24, no. 24, pp. 4337–4351, 2003.
View at: Publisher Site | Google Scholar
S. Van Vlierberghe, P. Dubruel, and E. Schacht, “Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review,” Biomacromolecules, vol. 12, no. 5, pp. 1387–1408, 2011.
View at: Publisher Site | Google Scholar
C. Vepari and D. L. Kaplan, “Silk as a biomaterial,” Progress in Polymer Science (Oxford), vol. 32, no. 8-9, pp. 991–1007, 2007.
View at: Publisher Site | Google Scholar
U. J. Kim, J. Park, C. Li, H. J. Jin, R. Valluzzi, and D. L. Kaplan, “Structure and properties of silk hydrogels,” Biomacromolecules, vol. 5, no. 3, pp. 786–792, 2004.
View at: Publisher Site | Google Scholar
X. Wang, J. A. Kluge, G. G. Leisk, and D. L. Kaplan, “Sonication-induced gelation of silk fibroin for cell encapsulation,” Biomaterials, vol. 29, no. 8, pp. 1054–1064, 2008.
View at: Publisher Site | Google Scholar
M. Fini, A. Motta, P. Torricelli et al., “The healing of confined critical size cancellous defects in the presence of silk fibroin hydrogel,” Biomaterials, vol. 26, no. 17, pp. 3527–3536, 2005.
View at: Publisher Site | Google Scholar
N. Guziewicz, A. Best, B. Perez-Ramirez, and D. L. Kaplan, “Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies,” Biomaterials, vol. 32, no. 10, pp. 2642–2650, 2011.
View at: Publisher Site | Google Scholar
B. B. Mandal, S. Kapoor, and S. C. Kundu, “Silk fibroin/polyacrylamide semi-interpenetrating network hydrogels forcontrolled drug release,” Biomaterials, vol. 30, no. 14, pp. 2826–2836, 2009.
View at: Publisher Site | Google Scholar
K. Numata, T. Katashima, and T. Sakai, “State of water, molecular structure, and cytotoxicity of silk hydrogels,” Biomacromolecules, vol. 12, no. 6, pp. 2137–2144, 2011.
View at: Publisher Site | Google Scholar
W. Tao, M. Li, and C. Zhao, “Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution,” International Journal of Biological Macromolecules, vol. 40, no. 5, pp. 472–478, 2007.
View at: Publisher Site | Google Scholar
Q. Fang, D. Chen, Z. Yang, and M. Li, “In vitro and in vivo research on using Antheraea pernyi silk fibroin as tissue engineering tendon scaffolds,” Materials Science and Engineering C, vol. 29, no. 5, pp. 1527–1534, 2009.
View at: Publisher Site | Google Scholar
N. Bhardwaj, S. Chakraborty, and S. C. Kundu, “Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering,” International Journal of Biological Macromolecules, vol. 49, no. 3, pp. 260–267, 2011.
View at: Publisher Site | Google Scholar
W. Xiao, J. He, J. W. Nichol et al., “Synthesis and characterization of photocrosslinkable gelatin and silk fibroin interpenetrating polymer network hydrogels,” Acta Biomaterialia, vol. 7, no. 6, pp. 2384–2393, 2011.
View at: Publisher Site | Google Scholar
X. Hu, Q. Lu, L. Sun et al., “Biomaterials from ultrasonication-induced silk fibroin-hyaluronic acid hydrogels,” Biomacromolecules, vol. 11, no. 11, pp. 3178–3188, 2010.
View at: Publisher Site | Google Scholar
M. K. Yoo, H. Y. Kweon, K. G. Lee, H. C. Lee, and C. S. Cho, “Preparation of semi-interpenetrating polymer networks composed of silk fibroin and poloxamer macromer,” International Journal of Biological Macromolecules, vol. 34, no. 4, pp. 263–270, 2004.
View at: Publisher Site | Google Scholar
G. M. Nogueira, M. A. De Moraes, A. C. D. Rodas, O. Z. Higa, and M. M. Beppu, “Hydrogels from silk fibroin metastable solution: formation and characterization from a biomaterial perspective,” Materials Science and Engineering C, vol. 31, no. 5, pp. 997–1001, 2011.
View at: Publisher Site | Google Scholar
F. Cilurzo, C. G. M. Gennari, F. Selmin, L. A. Marotta, P. Minghetti, and L. Montanari, “An investigation into silk fibroin conformation in composite materials intended for drug delivery,” International Journal of Pharmaceutics, vol. 414, no. 1-2, pp. 218–224, 2011.
View at: Publisher Site | Google Scholar
A. C. Sagle, H. Ju, B. D. Freeman, and M. M. Sharma, “PEG-based hydrogel membrane coatings,” Polymer, vol. 50, no. 3, pp. 756–766, 2009.
View at: Publisher Site | Google Scholar
J. L. Roam, H. Xu, P. K. Nguyen, and D. L. Elbert, “The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres,” Biomaterials, vol. 31, no. 33, pp. 8642–8650, 2010.
View at: Publisher Site | Google Scholar
M. Li, S. Lu, Z. Wu, K. Tan, N. Minoura, and S. Kuga, “Structure and properties of silk fibroin-poly(vinyl alcohol) gel,” International Journal of Biological Macromolecules, vol. 30, no. 2, pp. 89–94, 2002.
View at: Publisher Site | Google Scholar
H. J. Jin and D. L. Kaplan, “Mechanism of silk processing in insects and spiders,” Nature, vol. 424, no. 6952, pp. 1057–1061, 2003.
View at: Publisher Site | Google Scholar
A. Matsumoto, J. Chen, A. L. Collette et al., “Mechanisms of silk fibroin sol-gel transitions,” Journal of Physical Chemistry B, vol. 110, no. 43, pp. 21630–21638, 2006.
View at: Publisher Site | Google Scholar
M. Li, W. Tao, S. Lu, and S. Kuga, “Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking,” International Journal of Biological Macromolecules, vol. 32, no. 3–5, pp. 159–163, 2003.
View at: Publisher Site | Google Scholar
M. Li, W. Tao, S. Kuga, and Y. Nishiyama, “Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment,” Polymers for Advanced Technologies, vol. 14, no. 10, pp. 694–698, 2003.
View at: Google Scholar
D. W. Jones, Introduction to the Spectroscopy of Biological Polymers, Academic Press, New York, NY, USA, 1976.
W. K. Surewicz and H. H. Mantsch, “New insight into protein secondary structure from resolution-enhanced infrared spectra,” Biochimica et Biophysica Acta, vol. 952, no. 2, pp. 115–130, 1988.
View at: Google Scholar
X. Chen, Z. Shao, N. S. Marinkovic, L. M. Miller, P. Zhou, and M. R. Chance, “Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy,” Biophysical Chemistry, vol. 89, no. 1, pp. 25–34, 2001.
View at: Publisher Site | Google Scholar
P. Monti, G. Freddi, S. Sampaio, M. Tsukada, and P. Taddei, “Structure modifications induced in silk fibroin by enzymatic treatments. A Raman study,” Journal of Molecular Structure, vol. 744–747, pp. 685–690, 2005.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2012 Huijing Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

1739

Downloads

3774

Citations